During embryogenesis, the heart develops functional left-right (LR) asymmetries. Perturbation of cardiac LR asymmetry, or laterality, often leads to complex congenital heart defects. There is strong evidence that ciliated cells play a role in establishing cardiac laterality. Work from mouse and zebrafish models indicate a conserved group of embryonic 'LR cilia' generate an asymmetric fluid flow that is required for normal LR patterning of the vertebrate embryo. However, the mechanisms by which LR cilia generate LR information remain unclear. In zebrafish, asymmetric fluid flow is produced by a ciliated epithelium in an organ called Kupffer's vesicle (KV). Unlike other vertebrates, the cells that give rise to LR cilia in KV are accessible and can be studied in the zebrafish embryo. We are using zebrafish to characterize genes and mechanisms that control KV formation and heart asymmetry. Using both forward and reverse genetic screens, we have identified genes that implicate three new pathways in the regulation of LR ciliated cells in KV: 1) Rho kinase signaling, 2) cell polarity and 3) ion pump-mediated ion flux. The specific aims of this project are to characterize the role of these three pathways in establishing a functional KV and normal cardiac laterality. The long- term objective of this study is to advance our understanding of how the heart develops distinct left and right sides, and to provide candidate genes that may aid in diagnosis and treatment of human congenital heart defects. To achieve the goals of this project, we will analyze gene function using mutants, antisense morpholino gene knockdowns and small molecule inhibitors. We have developed a method to deliver morpholinos specifically to the KV cell lineage to analyze loss-of-function of these proteins specifically in LR ciliated cells. This approach allows us to distinguish the role of a particular gene in KV cells from its roles in other cell types in the embryo. To analyze LR ciliated cells in KV, we will take advantage of a unique set of techniques and tools available in zebrafish. These include real-time imaging of KV development in live embryos, immunostaining of KV cells with a collection of markers that reveal KV cellular architecture and videomicroscopy of asymmetric fluid flow. Results from this project will define genes and mechanisms that regulate LR cilia and heart laterality, and potentially provide insight into heart disease. PUBLIC HEALTH RELEVANCE: Congenital heart disease is the most common birth defect. Progress has been made in understanding heart defects, but for many cases the underlying cause is unknown. This project focuses on mechanisms that control heart development. Our goal is to identify genes that may aid in diagnosis, treatment or prevention of congenital heart disease.